Efficacy And Its Mechanism Of Preventing And Treating Early Subchondral Bone Lesions In Posttraumatic Osteoarthritis Secondary To Rabbit Acetabular Fractures By Icariin

Background:As the most common complication of acetabular fractures,Post-traumatic osteoarthritis(PTOA)is always treated with symptomatic relieving treatments at early stage or total hip arthroplasty at late stage.There is still no effective option to prevent or cure this kind of disease.Herba Epimedii is a regular herb in Traditional Chinese Medicine(TCM)over the millenniums.It is warm in nature,pungent and sweet in taste and belongs to the liver and kidney meridian.It has the effects of invigorating kidney and Yang,expelling wind and dampness,nourishing essence and bone.Over the long history of TCM development,Epimedium has long been used to treat bone and joint diseases,especially“Guwei”,or osteoposrsis.As the extractive of Herba Epimedii,Icarrin has multiple biological activities which include the potential application for the treatment of osteoporotic changes in subchondral bone at early stage of PTOA.In order to take advantage of TCM for preventing and treating PTOA following acetabular fractures,it is essential to reveal characteristics and pathogenesis of the early subchondral changes of the disease and analyzing the efficacy and mechanism of Icarrin at the present study.Part one Establishment and vertification of an animal model of posttraumatic osteoarthritis secondary to rabbit acetabular dorsal wall fractureObjective: To develop and evaluate an animal model of posttraumatic osteoarthritis following acetabular dorsal wall fractures for revealing the pathogenesis and further utilizing TCM to prevent and treat this kind of disease.Methods:A total of 36 mature male New Zealand white rabbits were equally divided into sham group(n=12),Non-ORIF group(n=12)and ORIF group(n=12).Except for the sham group,rabbits had survival surgeries to create acetabular fractures of dorsal wall for simulating dashboard impaction mechanism.The ORIF group received open reduction and internal fixation,while fractures in the Non-ORIF group were left as displaced and no fixation was applied,but accompanying transverse acetabular fracture and hip dislocation were reduced.Intraoperative appearance and postoperative recovery were recorded.Anteroposterior radiographs of the hips were took,then euthanization was conducted and the hips were harvested at 3 weeks,6 weeks and 6 months,respectively. Macroscopic,radiographic characteristics of the hips were recorded and assessed by a radiographic scoring scale,while Safranin O and fast green,H-E and Tuluidine blue staining were used to examine the cartilage tissue histopathological changes and assessed by OARSI Grading system at 3 weeks,6 weeks and 6 months,respectively.Results: Out of 24 modeled acetabula,21(87.5%)were pure dorsal wall fractures as proposed and showed a high consistency and reproducibility.Compared with the Sham group,the hips in the Non-ORIF group and ORIF group experienced progressive joint degeneration from 3 weeks.In the Non-ORIF group,there had been cartilage degeneration and joint space narrowing at 3 weeks,while obvious cartilage erosion,sclerotic subchondral bone and lots of osteophytes can be seen at 6 weeks.This kind of changes is consistent with clinical PTOA developmental features that would progress early and quickly.At 6 months,the Non-ORIF group demonstrated severe degeneration which exhibited dramatically malformed and hypertrophic joints accompanied by subluxation of the hip that is also in line with clinical late characteristics of PTOA following acetabular fractures.Compared with Non-ORIF group,although there was no significance in the semiquantitative scores,but ORIF showed significant lower OARSI grade at 6 months.The ORIF group kept much better histopathological morphological sctructure,better cartilage condition and showed improvements for the PTOA changes.Conclusion:The rabbit model,which was made by outlining fracture and joint impaction,with high consistency and reproducibility demonstrated early and quick posttraumatic osteoarthritic changes that is consistent with the features of clinical PTOA following acetabular fractures.The changes at 6 months of the model is also very similar to the late PTOA characteristics of human hips following acetabular fractures.It provides a new selection for exploring the pathological characteristics and pathogenesis of this kind of disease and must be useful for developing and evaluating TCM treatments to preventing and treating the disease.Part two Study on the characteristics and mechanism of early subchondral bone lesions in posttraumatic osteoarthritis secondary to rabbit acetabular dorsal wall fracturesObjective:To study on the characteristics and mechanism of early subchondral pathological changes of rabbit hip posttraumatic osteoarthritis following acetabular dorsal wall fractures.Methods: A total of 48 mature male New Zealand white rabbits were equally divided into sham group(n=16),Non-ORIF group(n=16)and ORIF group(n=16),and the right hips of rabbits in the sham group were considered as control group(n=16).Survival surgeries were conducted as part one.Anteroposterior radiographs of the hips and serums were collected,then euthanization was conducted and the hips were harvested at 3 days,3 weeks,6 weeks and 6 months,respectively.The microstructural changes of the acetabular subchondral bone were analyzed by Micro CT and the CTXI,P1 NP and BALP levels in serum were tested by ELISA.Rt-PCR and Western Blot were used to detect the gene and protein changes in the subchondral bone tissues.The differences of osteoclast activation and osteocyte apoptosis in subchondral bone between the groups were distinguished by TRAP staining and TUNEL,respectively.Results: In the Non-ORIF group of 3 weeks and 6 weeks,Micro CT showed significant lower Tb.BMD,BVF,Tb.Th and Ct.Th,and significant higher Tb.Sp and Ct.Po when compared with Sham group,that means conspicuous osteoporotic changes in the early sunchondral bone.In addition,significantly increaded CTXI concentration was simultaneously recorded from 3 days to 3 weeks in the Non-ORIF group,but amplication of CTXI was reduced at 6 weeks.While progressively significantly decreased P1 NP and BALP concentrations were recorded from 3 days to 6 weeks in the Non-ORIF group.Compared to the Control and Sham groups,Rt-PCR and Western Blot recorded increasing RANKL and less OPG expression at 3 days and 3 weeks,but the amplification of RANKL and decreasing amplitude of OPG were lessened at 6 weeks.More Trap positive multinuclear cells in subchondral bone in the Non-ORIF at 3 weeks when compared to the Sham group.At the same time,TUNEL showed more osteocytes apoptosis at 3 weeks,while Caspase3,Bax expression were significantly increased and Bcl-2expression was decreased in the Non-ORIF at 3 days and 3 weeks,but their amplitudes were all decreased at 6 weeks.Be important,osteocyte specific SOST gene and Sclerostin protein significant decreasing were also recorded at 3 days and 3 weeks in the Non-ORIF group.Compared to the Non-ORIF group,the ORIF group showed much better osteoporotic changes in the sunchondral bone,much higher Tb.Th,Tb.N and lower Tb.Sp at 3 weeks and much higer Tb.BMD,BVF,Tb.Th and lower Tb.Sp at 6 weeks were observed.Besides,less CTXI and more P1 NP and BALP were detected in the serum from 3 days to6 weeks.Meanwhile,Rt-PCR and Western Blot tested much less RANKL and more OPG expression at all three time points,that was closely associated with the decreased Trap positive multinuclear cells in subchondral bone at 3 weeks.Moreover,Ameliorative osteocyte specific SOST gene and Sclerostin protein expression were accompanied by less Caspase3,Bax and more Bcl-2 expression during the same intervals in the ORIF group.Conclusion:The differentiation and activation of osteoclasts and osteoporotic changes in early subchondral bone are considered to be the characteristics of PTOA changes following acetabular dorsal wall fractures and it is related to the apoptosis of osteocytes in the subchondral bone.Subchondral changes are closely related with the above cartilage degeneration that is conformed to the guidance of holism philosophy in TCM for pathological diagnosis.ORIF could attenuate osteocyte apoptosis and restrain the differentiation and activation of osteoclast by reducing and fixing the displaced intra-articular fractures in order to improve stress concentration so as to get a better prognosis of PTOA.Part three Efficacy and its mechanism of preventing and treating early subchondral bone lesions in posttraumatic osteoarthritis sencondary to rabbit acetabular fractures by Icariin Objective: To observe the efficacy of Icarrin in preventing and treating PTOA secondary to acetabular dorsal wall fractures and analyze its mechanism,so as to provide new ideas for the development and utilization of TCM to prevent and treat such disease.Methods:A total of 64 mature male New Zealand white rabbits were equally divided into sham group(n=16),Non-ORIF group(n=16),ORIF group(n=16)and ICA group(n=16),and the right hips of rabbits in the sham group were considered as control group(n=16).Survival surgeries were conducted as part one and rabbits in the ICA group were treated with icarrin(60mg/kg/day)by intragastric administration for consecutive 21 days.Anteroposterior radiographs of the hips and serums were collected,then euthanization was conducted and the hips were harvested at 3 days,3 weeks,6 weeks and 6 months,respectively.The microstructural changes of the acetabular subchondral bone were analyzed by Micro CT and the CTXI,P1 NP and BALP levels in serum were tested by ELISA.Safranin O and fast green,H-E and Tuluidine blue staining were used to examine the cartilage tissue histopathological changes and assessed by OARSI Grading system.Rt-PCR and Western Blot were used to detect the gene and protein changes in the subchondral bone tissues and the differences of osteoclast activation in subchondral bone between the groups were distinguished by TRAP staining.Results:Although there was no significant differences in histopathological OARSI grades in the ICA group when compared with the ORIF group at 6 months,but Micro CT showed much better improvements on the osteoporotic changes in the early sunchondral bone at 3 weeks and 6 weeks.Higher Tb.BMD,BVF,Tb.Th,Tb.N,Ct.Th and lower Tb.Sp,Ct.Po were recorded.Moreover,significant lower CTXI and higher P1 NP and BALP were simultaneously detected in the serum at all three time points in the ICA group.Corresponding,Rt-PCR and Western Blot tested much less RANKL and more OPG expression,which was tightly related to the further reduction of Trap positive multinuclear cells in subchondral bone during the same interval.Besides,Rt-PCR and Western Blot also recorded further less Caspase3,Bax and more Bcl-2 expression in ICA group at the same time when compared with the ORIF group,accompanied by further amelioration of osteocyte specific SOST gene and Sclerostin protein expression.Noteworthy,there were already obvious subchondral sclerotic changes in the ORIF group at 6 months,while sclerotic transformation got much more relieves and significant lower Tb.BMD,BVF,Tb.Th,Ct.Th were recorded in the ICA group at the same time.Conclusion: Icarrin could improve the microstructural changes in subchondral bone in order to preventing early subchondral pathological changes of PTOA secondary to rabbit acetabular dorsal wall fractures,which reflects the guidance of holism philosophy in TCM during treating PTOA following acetabular fractures,or the improvement of sunchondral bone pathological changes is beneficial to reduce the degeneration of articular cartilage and could achieve a coordinated efficacy between local lesions and holistic health.Icariin could not only inhibit the differentiation and activation of osteoclast,but also significantly enhance the osteogenic activity.The efficacy of Icarrin is considered to be related with suppressing the osteocyte apoptosis,but further research is needed to explore their potential mechanism.